RESEARCH ARTICLE Open Access

Secondary resistance to immunotherapyassociated with β-catenin pathwayactivation or PTEN loss in metastaticmelanomaJonathan A. Trujillo1 , Jason J. Luke1, Yuanyuan Zha1, Jeremy P. Segal2, Lauren L. Ritterhouse2, Stefani Spranger2,3,Karen Matijevich1 and Thomas F. Gajewski1,2*

Abstract

Background: While cancer immunotherapies including checkpoint blockade antibodies, adoptive T cell therapy,and even some vaccines have given rise to major clinical responses with durability in many cases, a subset ofpatients who initially respond subsequently develop secondary resistance to therapy. Tumor-intrinsic mechanismsof acquired immunotherapy resistance are incompletely understood.

Methods: Baseline and treatment-resistant tumors underwent molecular analysis via transcriptional profiling orgenomic sequencing for oncogenic alterations and histologic analysis for T cell infiltration to investigate mechanismscontributing to T cell exclusion and acquired resistance to immunotherapy.

Results: We describe two patients with metastatic melanoma who initially showed a durable partial response to eithera melanoma-peptide/interleukin-12 vaccine or combined anti-CTLA-4 + anti-PD-1 therapy, but subsequently developednew treatment-resistant metastases. In the first case, the recurrent tumor showed new robust tumor expression of β-catenin, whereas in the second case genomic sequencing revealed acquired PTEN loss. Both cases were associatedwith loss of T cell infiltration, and both pathways have been mechanistically linked to immune resistance preclinically.

Conclusion: Our results suggest that secondary resistance to immunotherapies can arise upon selection for newoncogenic variants that mediate T cell exclusion. To identify the spectrum of underlying mechanisms of therapeuticresistance, similar evaluation for the emergence of tumor-intrinsic alterations in resistant lesions should be doneprospectively at the time of relapse in a range of additional patients developing secondary resistance.

Keywords: Immune checkpoint blockade, Peptide vaccine, Secondary resistance, PTEN-loss, β-Catenin activation,Immune exclusion, Next-generation sequencing

BackgroundApproximately 22–60% of patients with metastatic mela-noma who have objective responses to immunotherapiessuch as anti-PD-1 and/or anti-CTLA-4 antibodies will sub-sequently relapse [1–4]. Mechanisms of immune-resistantcancer progression in this context are incompletely under-stood. While a significant focus has been placed on

interrogating baseline tumor biopsies for genomic andimmune determinants of primary resistance, longitudinalanalysis of tumors at disease progression are needed touncover molecular drivers of secondary resistance.Several cases of secondary resistance to immunother-

apies have been reported that revealed tumor cell-intrinsicdefects in antigen processing/presentation [5–9] and inIFN-γ signaling [10–12]. Early studies found evidence thatmelanoma patients who initially responded to cytokinesand adoptive T cell-based therapies developed secondaryresistance through cancer cell loss of beta-2 microglobulin(B2M), the subunit necessary for antigen presentation by

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: tgajewsk@medicine.bsd.uchicago.edu1Department of Medicine, University of Chicago, 5841 S. Maryland Ave,MC2115, Chicago, IL 60637, USA2Department of Pathology, University of Chicago, 5841 S. Maryland Ave,MC2115, Chicago, IL 60637, USAFull list of author information is available at the end of the article

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MHC class I molecules [5]. Analysis of longitudinal tumorbiopsy specimens from metastatic melanoma patientstreated with anti-CTLA-4 or anti-PD-1 identified a subsetof initial responders whose disease progressed with resist-ant tumors no longer expressing B2M [6]. Recently,acquired B2M loss also has been identified in a metastaticmelanoma patient with secondary resistance to PD-1blockade [7], in a case of lung cancer that developedresistance to the anti-CTLA-4 + anti-PD-1 combination[8], and in resistant brain metastases in two patients withmismatch-repair deficient colorectal cancer that acquiredresistance to anti-PD-1 therapy [13]. Defective IFN-γsignaling, such as through inactivating mutations in Januskinases (JAK1 or JAK2) or in the interferon-gamma recep-tor 1 (IFNGR1), has also been proposed to correlate withresistance to anti-PD-1 therapy [7, 11, 12]. Genome-scaleCRISPR-Cas9 mutagenesis screens of cancer cells haveprovided evidence for a causal relationship between de-fects in antigen processing and presentation machinery inpromoting resistance to T cell-based immunotherapies[14, 15]. Thus, loss of B2M and defective IFN-γ signalingcan contribute to a T cell-resistant phenotype and aretumor-intrinsic determinants of resistance to immuno-therapies. However, such defects are not found in alltumors, and these escape mechanisms are difficult to drugtherapeutically. Thus, continued analysis of secondaryresistance samples is important, in the hope of identifyingpathways that might be amenable to future therapeuticintervention.Molecular analysis of baseline tumor biopsies has re-

vealed that selected oncogenic alterations in tumor cellscan promote immune cell exclusion from the tumormicroenvironment and may contribute to primary im-munotherapy resistance. In pre-clinical studies, tumorcell-intrinsic activation of the Wnt/β-catenin pathwayhas been identified to mediate T cell exclusion from thetumor microenvironment and primary resistance to im-mune checkpoint blockade therapy [16]. Mechanisticstudies using a genetically engineered mouse model ofmelanoma revealed that β-catenin activation resulted in aloss of BATF3-lineage dendritic cells in the tumor micro-environment, leading to failure of T cell priming and lackof T cell accumulation in tumors. Adoptively transferredtumor-specific T cells or prophylactic vaccination aimed atinducing endogenous anti-tumor memory CD8+ T cellsalso failed to control β-catenin-expressing tumors in thismodel, due to defective effector T cell trafficking [17].These data demonstrated that tumor cell-intrinsic β-catenin activation confers an immune-resistant phenotypethat impairs immune control even in the face of therapeu-tically induced anti-tumor T cells. These findings raise thepossibility that tumor recrudescence could occur as aconsequence of upregulation of β-catenin by cancer cells,resulting in secondary resistance to immunotherapy.

Beyond β-catenin, gene deletions and loss-of-function mu-tations of the tumor suppressor phosphatase and tensinhomolog (PTEN) have also been associated with poor Tcell infiltration in the tumor microenvironment inmetastatic melanoma [18]. Loss of PTEN, which leadsto increased activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, has been associated withprimary resistance to PD-1 blockade in melanoma[18]. Whether acquired PTEN loss leads to secondaryimmune resistance to immune checkpoint therapies inmelanoma has not been reported.In this context, we describe two patients who initially

showed a durable partial response to immunotherapy yetsubsequently developed new treatment-resistant metas-tases. Both cases showed loss of a T cell-inflamed tumormicroenvironment, providing an opportunity to investi-gate potential molecular aberrations associated with lossof T cell infiltration and immunotherapy resistance.

MethodsImmunohistochemistryImmunohistochemistry (IHC) for S-100, Melan-A andHMB-45 and the respective controls were performed onformalin-fixed, paraffin-embedded (FFPE) tissue sectionsby the Clinical Hematology and ImmunohistochemistryLaboratories of the University of Chicago Hospitals.Stained IHC samples were evaluated by clinical patholo-gists at the University of Chicago Hospitals. CD8 and β-catenin immunohistochemistry staining was performed bythe Human Tissue Resource Center (HTRC) at the Univer-sity of Chicago. Immunohistochemistry staining wasperformed using a CD8-specific monoclonal antibody (Ab,CD8 clone C8/144B, R&D Systems) and a β-cateninmonoclonal Ab (clone CAT-5H1, Life Technologies) incombination with a secondary goat anti-mouse immuno-globulin G (IgG) conjugated to an alkaline phosphatase(Biocare Medical). Slides were scanned using a CRiPanoramic Scan Whole Slide Scanner and viewed withPanoramic Viewer 1.15.4 (3DHISTECH).

Multiplex immunofluorescenceMultiplex immunofluorescence (IF) was done accordingto Opal kit (Perkin Elmer) instruction. IF staining wasperformed using PTEN Ab (clone 6H2.1, EMD), CD8Ab (clone C8/144B, R&D Systems) and Sox10 Ab (clone20B7, R&D Systems). Briefly, FFPE tissue sections werebaked for 1 h at 65 °C, cleared by submerging in histo-clear solution (Fisher) for 10 min three times. Thesections were then rehydrated by submerging in 100, 95,and 75% ethanol solutions, rinsed in distilled water, andfixed in 10% normal buffered formalin solution for 20min. After rinsing in water, the slides were placed inEDTA (pH 9) buffer. Antigen retrieval was performed inTintoRetriever Pressure cooker at 115 °C for 20 min.

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The tissue sections were then blocked with properblocking buffer, incubated with PTEN Ab for 1 h atroom temperature, washed three times in Tris-bufferedsaline with Tween 20 (TBST) buffer (pH 7.6), incubatedwith HRP-conjugated secondary Ab, followed by threewashes in TBST, and incubated with proper Opalreagent for 10 min at room temperature. The procedurewas then repeated for CD8 and Sox10. After all thetargets were labeled, the sections were incubated withDAPI solution for 5 min at room temperature, andmounted in ProLong Diamond Antifade Mountant(Invitrogen). The tissue sections were then scannedusing Vectra Polaris (Perkin Elmer) and the images werecaptured using Phenochart (Perkin Elmer).

Gene expression profilingSamples were obtained from eligible patients who signedwritten informed consent for clinical trials and tissuebiobanking at the University of Chicago. Core biopsieswere obtained from material resected from patients aspart of standard clinical management. Tumor wasgrossly isolated from surrounding normal tissue and asmall piece of tumor was snap frozen in liquid nitrogen.RNA was later isolated from cryopreserved tumor biopsyusing Allprep DNA/RNA mini kit (Qiagen, Inc) andquality controlled by the Human Immunologic Monitor-ing Facility at the University of Chicago. The transcrip-tion profiling was done using the Human Genome U133plus 2.0 Array (Affymetrix) at the Genomic Core Facilityat the University of Chicago. Subsequent data analysisinvolved global normalization of array values to themedian signal intensity of all genes on the array. The geneexpression values are log2-transformed.

In vitro T cell priming and ELISpotHeparinized blood was drawn before treatment, monthly ontreatment, and at the end of the vaccine study. The four pep-tides used in the vaccine include: Melan-A (AAGIGILTV),gp100 (KTWGQYWQV), MAGE-3 (FLWGPRALV), andNA17 (VLPDVFIRCV). Peripheral blood mononuclear cells(PBMCs) were isolated using Ficoll-Hypaque gradient centri-fugation and cryopreserved in liquid nitrogen freezer vaporphase. Antigen-specific CD8+ T cells were expanded throughan in vitro stimulation step. Briefly, the PBMCs were thawed.CD8+ cells were isolated using CD8 microbeads (MiltenyiBiotech). The flow through CD8-negative cells were pulsedwith 50 μM peptide (either derived from Epstein-Barr virus[EBV; GLCTLVAML), Melan-A (AAGIGILTV), gp100 (KTWGQYWQV), MAGE-3 (FLWGPRALV), or NA17 (VLPDVFIRCV)] in the presence of 2.5 μg/ml beta-2 microglobu-lin for 1 h at 37 °C. The peptide pulsed CD8-negative cellswere then washed and irradiated with a total dose of 3000rad and co-culture with CD8+ cells at a 5:1 ratio for 5 days at37 °C. Recombinant human IL-2 (rhIL-2) at a concentration

of 20 units/ml was added into the culture on day 2. On day5, CD8+ cells were collected and co-cultured with irradiated,peptide pulsed CD8-negative cells and rhIL-2 for another 5days. On day 10, expanded CD8+ cells were collected andseeded on the ELISpot plate pre-coated with IFN-γ Ab(clone 1-D1K, Mabtech, Inc) and co-cultured with peptide-pulsed T2 cells overnight. On the following day, the platewas washed and incubated with a biotinylated anti-IFN-γsecondary Ab (clone 7-B6–1, Mabtech, Inc.) for 2 h at roomtemperature. Following three washes, the plate was incubatedwith streptavidin-conjugated AP for 1 h, washed, and incu-bated with AP substrate. Excess substrate was removed byrinsing with tap water. The plate was then air-dried,captured, and counted using a CTL-ImmunoSpot S6 CoreAnalyzer (Cellular Technology Ltd). All samples wereanalyzed in triplicate.

Next-generation genomic sequencingNext-generation genomic sequencing (NGS) was per-formed using the OncoScreen ST2.0 or OncoPlus, the Uni-versity of Chicago Clinical Laboratory ImprovementAmendments-certified next-generation sequencing plat-forms [19]. The OncoScreen ST2.0 clinical assay was per-formed on tissue derived from the wide local excision scalpmelanoma. OncoSreen ST2.0 is a 50-gene solid tumorpanel that uses the Ion Ampliseq Cancer Hotspot Panel V2primer set (Thermo Fisher Scientific) for amplification of207 hot-spot targeted amplicons across 50 genes [19]. TheOncoScreen ST2.0 platform includes the genes listed inAdditional file 1: Table S1.Tissue slides and blocks were reviewed by a pathologist

to select the appropriate material for NGS testing. DNAwas isolated from micro-dissected FFPE tumor tissueusing the QIAamp DNA FFPE Tissue Kit (Qiagen). Fol-lowing extraction, DNA was quantified using the Qubitfluorometric assay (Thermo Fisher Scientific) and furtherassessed for quantity and quality using a quantitative PCRassay (hgDNA Quantitation and QC kit, KAPA Biosys-tems). FFPE DNA was amplified for somatic mutations lo-cated within mutational hotspot regions of 50 cancer-related genes using multiplex PCR reagents (ThermoFisher Scientific). PCR products were quantitated usingthe Qubit assay then used as a substrate for NGS librarypreparation (HTP Library Preparation Kit, KAPA Biosys-tems), using selected patient-specific adapter index se-quences. Libraries were quantified using a quantitativePCR assay (Library Quantification Kit, KAPA Biosystems),then pooled and sequenced via the Illumina MiSeq system(2 × 152 bp paired-end sequencing). Sequencing data wasanalyzed via custom-designed bioinformatics pipelines onan University of Chicago HIPAA compliant high perform-ance computing system, using the hg19 (GRCh37) humangenome reference sequence for alignment [19]. Limit ofdetection: 5% mutant alleles.

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The resistant cerebellar metastases underwent next-generation genomic sequencing using the OncoPlusassay, a clinically validated hybrid capture genomic se-quencing platform comprising 1212 commonly alteredcancer genes for mutational and copy number analysis(genes listed in Additional file 1: Table S2) [19].A pathologist reviewed the original pathology report, ex-

amined candidate H&E stained slides, and selected the ap-propriate block for NGS testing. DNA was isolated frommicrodissected FFPE tumor tissue using the QiaAMP DNAFFPE Tissue Kit (Qiagen). Following extraction, DNA wasquantified using the Qubit fluorometric assay (ThermoFisher Scientific) and further assessed for quantity andquality using a quantitative PCR assay (hgDNA Quantita-tion and QC kit, KAPA Biosystems). DNA was subjected toultrasonic fragmentation and subsequent library prepar-ation using adapter molecules containing patient-specificindex sequences (HTP LibraryPreparation Kit, Kapa Biosys-tems). After library amplification, quantification and pool-ing, fragments originating from targeted genomic regionswere enriched using a panel of biotinylated oligonucleotides(SeqCap EZ, Roche Nimblegen) supplemented with add-itional oligonucleotides (xGen Lockdown Probes, IDT).After subsequent amplification and pooled library quantifi-cation, libraries were sequenced in rapid run mode on aHiSeq 2500 system (Illumina) to produce 2 × 101 bp pairedend sequencing reads. Sequencing data was analyzed viacustom-designed bioinformatics pipelines on an Universityof Chicago HIPAA compliant high performance computingsystem, using the hg19 (GRCh37) human genome referencesequence for alignment. Limit of detection: For mutations,insertions and deletions, limit of detection is 10% mutantalleles (roughly corresponding to 20% tumor cells). Limit ofdetection for fusions/translocations is 20% tumor cells.Gene fusions cannot be detected in the rare occurrence ofa fusion between ALK, RET or ROS1 and a partner geneless than 100,000 bp distant. Limit of detection for copynumber changes is >4X or < 0.5X normal copy number,with relevant equivocal changes reported at >2X or < 0.6X.

ResultsSecondary immune resistance associated with β-cateninactivationA 54-year-old Caucasian male with metastatic melanoma(diagnosed prior to the era of B-Raf inhibitors) initially re-ceived interleukin-2 without benefit followed by right hep-atic lobectomy. Two years following surgery pulmonarymetastases were observed leading to mediastinoscopy withbiopsy of right level 4, lower paratracheal lymph node.Pathology was consistent with metastatic melanoma withtumor cells extensively immunoreactive for HMB-45 andfocally immunoreactive for Melan-A and S-100. The pa-tient was HLA-A2-positive and enrolled on a clinical trialof a multi-peptide vaccine combined with interleukin-12

[20]. The patient was treated every 3 weeks for one yearand a durable partial response was observed (RECIST 1.0)[20]. The patient was followed by close observation by ser-ial computed tomography scans until a new metastaticlesion in the pelvis was confirmed by biopsy approximately3 years later.A biopsy was performed of the new lesion, both to con-

firm recurrent melanoma and to study the immunobiologyof the tumor microenvironment relative to that of the pre-treatment tumor. Immunohistochemical studies showedextensive staining by HMB-45 and focal immunoreactivityfor Melan-A and S100, confirming melanoma and expres-sion of these two antigens in the new lesion. Analysis of thepretreatment biopsy revealed strong and homogenousCD8+ T cell infiltration (Fig. 1a upper left panel). Consist-ent with immunohistochemistry analysis, gene expressionprofiling revealed evidence for a T cell-inflamed tumormicroenvironment including T cell markers, chemokines,and interferon-induced genes (Fig. 1b). In contrast, immu-nohistochemical staining of the recurrent tumor showedabsence of infiltrating CD8+ T cells (Fig. 1a lower leftpanel). Gene expression profiling revealed markedly re-duced chemokines and other immune genes compared tothe original tumor biopsy (Fig. 1b), consistent with selectionfor a microenvironment that failed to recruit T cells. Basedon the ability of activated β-catenin to mediate T cell exclu-sion [16], stabilized β-catenin was analyzed by immunohis-tochemistry. Strikingly, the pre-treatment sample hadminimal staining for β-catenin, whereas the recurrenttumor showed strong staining that included nuclearlocalization (Fig. 1a right panels). Expression of four definedβ-catenin target genes and also of β-catenin transcriptswere upregulated in the recurrent tumor (Fig. 1c). Thus,the immune resistance phenotype exhibited by the new me-tastases was associated with β-catenin pathway activation.Expression of three of the four antigens targeted by the vac-cine (Melan-A, MAGE-3, gp100) was detected in the pre-treatment tumor specimen by gene-expression microarrayanalysis (Fig. 2a). Retained expression of tumor antigenstargeted by the vaccine was assessed by gene-expressionmicroarray analysis, and Melan-A, MAGE-3, and gp100were all confirmed to be expressed by the recurrent tumor(Fig. 2a). Analysis of peripheral blood indicated an increasein T cell reactivity against all four peptides used in the vac-cine, Melan-A (AAGIGILTV), gp100 (KTWGQYWQV),MAGE-3 (FLWGPRALV), and NA-17 (VLPDVFIRCV)during initial treatment (Fig. 2b). Re-analysis of the Tcell responses from peripheral blood obtained at thetime of progression revealed persistent reactivityagainst three of the peptides (gp100, Melan-A andMAGE-3), consistent with T cell memory against atleast these three epitopes (Fig. 2c). The patient wassubsequently treated with dacarbazine chemotherapy,which resulted in a partial response.

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Secondary immune resistance associated with biallelicPTEN lossA 23-year-old Asian male with metastatic BRAF-V600Emelanoma was originally treated with B-Raf inhibitor +MEK inhibitor (trametinib and dabrafenib) and palliativeradiation to a sacral metastasis. The patient had a mixedresponse to therapy, and was subsequently treated withcombination anti-CTLA-4 + anti-PD-1 therapy with ipili-mumab and nivolumab according to the FDA-approveddose and schedule. The patient achieved a durable partialresponse to therapy. Eight months later, the patient devel-oped a left midclavicular nodule that was biopsied and

confirmed to be metastatic melanoma and subsequentlytreated with radiation. The patient continued therapy withnivolumab for a total of fourteen months until imagingdemonstrated early evidence of disease progressionprompting reinduction with ipilimumab + nivolumab.After eighteen months total on immune checkpoint block-ade, the patient developed multi-site disease progression,including new osseous lesions, mediastinal and hilarlymphadenopathy, and a cerebellar tumor. The patientunderwent a craniotomy and resection of the cerebellartumor that confirmed metastatic melanoma. The patientultimately received palliative radiation and ultimately died

Fig. 1 Tumor gene expression profiling, CD8+ T cell infiltration, and β-catenin status at baseline and at recurrence. a Immunohistochemistrystaining for CD8 (red staining) and β-catenin (red staining), in baseline (pre-treatment, right lower paratracheal lymph node metastasis) andrecurrent (treatment-resistant, left inguinal lymph node metastasis) melanoma tumor biopsies. b Expression level of immune-related genes inbaseline and recurrent tumor samples measured by genome expression microarray. Depicted are the genes GZMK, CD8A, CCL4, CXCL9, CCL3,CCL5, HLADMA, CXCL10, TRGC2, TRAA, NKG7, CD2, TRGV9, TRGC2, PRF1, CD8B, TRBC1, CD38, IL1R2, IL23A, TRBC1, IL2RG, CCL18, CD27, IFNG, RAC2,TNFSF10, CD3E, TAP1, TNFRSF9, HLADPA1, TAP2, NLRP1, STAT1, CXCL13. Genes in bold font are being shown in red and were previously part of ourcore signature associated with CD8+ T cells [21]. c Gene expression levels of six β-catenin target genes (VEGFA, TCF12, MYC, TCF1, EFNB3, APC2) aswell as β-catenin (CTNNB1, red) itself. Genome microarray data (b and c): expression levels for each gene transcript are normalized to mediansignal intensity of all genes on the microarray, and represented as normalized hybridization intensity data and expressed as expression units

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with progressive metastatic disease. To explore mecha-nisms of immunotherapy resistance, pre-treatment andtreatment-resistant tumor biopsies were analyzed forsomatic genetic abnormalities.Tumors were analyzed by next-generation genomic

sequencing (NGS) using a clinically validated amplicon-based assay (OncoScreen ST2.0) or a hybrid capturegenomic sequencing platform (OncoPlus), respectively,comprising a panel of commonly altered cancer genes formutational and copy number analysis (Fig. 3 and Table 1).The pathogenic variants detected in the pre-treatmentscalp melanoma included the BRAF-V600E mutation(BRAF c.1799 T > A, p.V600E), amplification of BRAF lo-cated on chromosome 7q34, and loss of the tumor sup-pressor gene CDKN2A located on chromosome 9p21.3(Fig. 3a). The treatment-resistant cerebellar metastasisalso had the same BRAF-V600E mutation (BRAF c.1799T > A, p.V600E), loss of CDKN2A, and BRAF amplifica-tion, but additionally demonstrated biallelic loss of thetumor suppressor gene PTEN located on chromosome10q23.31 (Fig. 3b). Both the pre-treatment and resistanttumors shared the BRAF amplification, which has beensuggested to confer relative resistance to BRAF inhibitortreatment [22]. Loss of CDKN2A has been suggested to co-operate with PTEN deletion to drive resistance to BRAF in-hibitors [23]. The treatment-resistant metastasis uniquelyharbored biallelic PTEN loss, while the pre-treatment bi-opsy had no detectable PTEN alterations. No mutationswere observed in the gene encoding beta-2-microglobulin(B2M), the required subunit necessary for surface expres-sion of the MHC class I molecule, or the gene encodinginterferon-receptor-associated Janus kinase 2 (JAK2) in ei-ther of the tumor specimens. No mutations conferringmicrosatellite instability were observed in the pre-treatmentor treatment-resistant tumors. Additional somatic alter-ations and copy-number events (Table 1) identified in thetreatment-resistant tumor were of uncertain significance.To determine whether genetic alterations in PTEN led

to loss of protein expression, multiplex immunofluores-cence was performed on the on-treatment midclaviculartumor biopsy during disease control and on thetreatment-resistant cerebellar tumor biopsy (Fig. 4).Histologic analysis of the biopsies revealed extensiveexpression of Sox10 identifying melanoma tumor tissue.While PTEN protein was expressed throughout the firstbiopsy during tumor control, it was absent from thesecond lesion that represented disease progression fol-lowing nivolumab + ipilimumab. The treatment-resistantbiopsy also revealed minimal staining for CD8+ T cellscompared to the earlier biopsy (Fig. 4), and was add-itionally associated with loss of stainable PTEN protein.These results were noteworthy based on prior mechanis-tic data indicating immunotherapy resistance uponPTEN loss [18].

Fig. 2 Peripheral tumor-reactive T cells persist at the time ofprogression. a Expression-level of targeted tumor antigens in pre-treatment (unfilled bars) and resistant (filled bars) tumor samplesdetermined by genome expression microarray (NA-17 not representedon the gene array). Gene expression of targeted antigen transcripts arenormalized to median signal intensity of all genes on the array andexpressed as expression units. b IFN-γ ELISpot assessing the T cellreactivity against the four peptides used in the vaccine, gp100, Melan-A, MAGE-3, NA-17, over time during initial treatment. PBMCs isolated ateach time point and stimulated with the indicated melanoma peptidesor media control. Samples analyzed in triplicate and presented as themean number of spots per number of PBMCs with standard deviation.Mean number of spots for each peptide compared to media control.P-values for gp100, Melan-A, MAGE-3, and NA17 peptide versus mediacontrol listed from top to bottom, respectively, at each time point,*p < 0.05, **p < 0.001, ***p < 000.1 (c) IFN-γ ELISpot showing persistentT cell reactivity against three melanoma peptides (gp100, Melan-A andMAGE-3) at the time of progression. PBMCs stimulated with mediacontrol, EBV antigen (control peptide), gp100, Melan-A, MAGE-3, andNA17 peptide. Samples analyzed in triplicate and presented as themean number of spots per number of PBMCs with standard deviation.Mean number of spots compared to media control. *p < 0.05,**p < 0.001, ***p < 000.1

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DiscussionImmunotherapeutic interventions, including checkpointblockade, adoptive T cell transfer, and some vaccinationapproaches have been developed as potent strategies to in-duce and enhance anti-tumor immunity, translating intoclinical efficacy in multiple tumor types [24]. Translationalstudies of anti-PD-1 antibodies and also experimentalvaccines have provided evidence that clinical response is

favored when CD8+ T cells are present within the tumormicroenvironment at baseline [25]. Absence of recruit-ment and activation of tumor antigen-specific T cells inthe tumor microenvironment has therefore been corre-lated with primary resistance to anti-PD-1 immunother-apy [26]. Two oncogenic events linked to poor T cellinfiltration and primary immunotherapy resistance aretumor cell-intrinsic β-catenin pathway activation and also

Fig. 3 Acquired genetic loss of PTEN in a therapy-resistant melanoma tumor in a patient previously responding to ant-CTLA-4 and anti-PD-1therapy. a and b Copy number alterations. Next generation sequencing of pre-treatment and therapeutic-resistant melanoma lesions showsacquired loss of PTEN in the treatment resistant tumor specimen but not the pre-treatment lesion. Log2 of fold- changes in the (a) pre-treatmenttumor sample (upper panel) and (b) treatment-resistant metastasis (lower panel). The analysis shows copy number changes in BRAF, PTEN,CDKN2A, FANCA, H3F3A, NOTCH1, PALB2, RAD51, RET, TSC1, TSC2. Copy number alterations are indicated in red. Genomic regions across thechromosomes that have no detectable alterations are indicated in blue or gray. Dotted lines indicate the Log2 fold-change cutoffs

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PTEN loss-of-function mutation or deletion [16, 18]. How-ever, whether secondary resistance might arise throughacquisition of tumor cell-intrinsic oncogenic alterations hadnot been known. Our current data provide evidence thatacquisition of active β-catenin signaling in tumor cells orPTEN loss might mediate secondary resistance to immuno-therapy even in the presence of circulating memory CD8+

T cells specific for tumor-expressed antigens.Our results suggest that a broad net should be cast

when evaluating for mechanisms of secondary resistanceto immunotherapy in patients. Recent data have revealedthat loss-of-function defects in beta-2 microglobulin andalso Jak signaling can be found in tumors that progressfollowing initial clinical response to anti-PD-1 [7]. Ourcurrent results argue that active immune exclusion mech-anisms of resistance also can occur, as in the β-cateninprotein stabilization identified in the vaccine-treatedpatient. There was not sufficient tissue obtained in the re-current tumor biopsy for exome or genomic sequencingto elucidate the mechanism of β-catenin activation in thiscase, but our previous results have demonstrated that β-catenin pathway activation in melanoma can be driven byactivating mutations in CTNNB1 (β-catenin) itself, inacti-vating mutations in inhibitors of β-catenin such as

AXIN1, or over-expression of specific Wnt ligands orFrizzled receptors [16]. Alterations that lead to Wnt/β-ca-tenin pathway activation are recurrent in melanoma [16,27] and other tumor types [28] and are associated with alack of T cell infiltration at baseline; however, clinicalimmunotherapy-specific outcome data, especially forimmune-checkpoint inhibitors, are still lacking. Thecurrent patient developed a β-catenin-expressing tumorvariant associated with immune escape. Immune surveil-lance and long-term protection against re-emergingcancer cells depends upon retention of tumor antigensand the presence of tumor-specific T cells. In this case, ac-quired immune resistance was not associated with loss ofexpression of melanoma antigens by the new metastasisnor linked to an absence of melanoma-specific T cellsfrom the immune repertoire. Rather, T cells failed to accu-mulate in the new resistant tumor in spite of the presenceof circulating memory T cells specific to three of the mel-anoma epitopes targeted by the peptide vaccine. It is ofinterest that he subsequently responded to chemotherapy,which suggests that the mechanisms of resistance withimmunotherapy versus chemotherapy may be distinct.The patient who developed therapeutic resistance to the

anti-CTLA-4 + anti-PD-1 combination progressed withmultisite disease including an immune-resistant brain me-tastasis. The near complete absence of CD8+ T cells fromthe resected brain tumor lesion supports immune exclu-sion as the putative resistance mechanism, and PTEN lossmay have contributed to ineffective CD8+ T cell accumu-lation. While the blood-brain-barrier regulates T cell traf-ficking into central nervous system tissue, it does notappear to be a major determinant of therapeutic resistanceto immune checkpoint inhibitors based on the high rateof efficacy observed against melanoma metastatic to thebrain [29, 30]. For example, intracranial responses to brainmetastases have been observed in 57% of patients, includ-ing a 26% complete response rate to previously untreatedintracranial lesions in melanoma patients treated withcombined nivolumab and ipilimumab [29]. Loss of PTENexpression has been correlated with shorter time to brainmetastases and reduced overall survival among patientswith BRAFV600-mutant melanoma implicating the PI3K-AKT pathway in the establishment of brain metastasis[31]. Thus, the functional interaction between mutatedBRAF and PTEN loss/PI3K-AKT activation in the currentpatient may have promoted brain metastasis and immuno-therapy resistance. Concurrent biopsies of extracranialmetastases were not clinically indicated and thus not per-formed in this patient, so we cannot rule out that distinctmechanisms apart from PTEN loss might be linked to re-sistant metastases in other anatomic sites. An analysis ofthe melanoma Cancer Genome Atlas (TCGA) data setfound that the frequency of deletions and loss-of-functionmutations in PTEN were greater in non-T-cell inflamed

Table 1 Genetic variants detected via next-generationsequencing of pre-treatment and treatment-resistant tumorspecimens

Baseline (pre-treatment) biopsy Treatment-resistant biopsy

Variants detected (NGS platform:OncoScreen ST2.0)

Variants detected (NGSplatform: OncoPlus)

Pathogenic variants Pathogenic variants

BRAF c.1799 T > A, p.V600E BRAF c.1799 T > A, p.V600E

CDKN2A loss CDKN2A loss

BRAF amplification BRAF amplification

PTEN loss

Variants of indeterminateclinical significance

APC c.385G > C, p.E129Q

APC c.6211A > G, p.I2071V

PTCH1 c.3641C > T, p.T1214 M

RET c.2939 + 6C > T

FANCA Loss

H3F3A Loss

NOTCH1 Loss

PALB2 Loss Equivocal

RAD51 Loss Equivocal

RET Loss

TSC1 Loss Equivocal

TSC2 Loss Equivocal

Abbreviations: NGS next-generation genomic sequencing, c. cDNA alteration,p., protein alteration

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tumors [18]. In addition, the absence of PTEN protein intumor samples correlated with diminished CD8+ T cell in-filtration and inferior outcomes to anti-PD1 in melanomapatients [18]. While an elevated frequency of PTENalterations has been specifically noted in melanoma brainmetastases [31], combination checkpoint blockade cangenerate a high response rate in brain metastases [29, 30],arguing that it remains immunotherapy responsive in amajor subset of cases. A prior study had reported thatPTEN alterations were not correlated with an immunegene signature in brain metastases, although this analysiswas not done in conjunction with clinical response [32].Consistent with our results, biallelic loss of PTEN wasexclusively identified in a treatment-resistant extracranialmetastasis from a patient with metastatic uterine sarcomawho achieved a durable complete remission with anti-PD-

1 therapy following resection of the sole immune-escapetumor [33].The possibility of activation of specific oncogene path-

ways in immunotherapy-resistant tumors raises the poten-tial for developing pharmacologic inhibitors of suchpathways towards restoration of T cell infiltration and im-munotherapy efficacy. There is renewed interest in develop-ing inhibitors of Wnt/β-catenin signaling that might bemore selective for the immune regulatory functions of thispathway. In addition, because PTEN loss-of-function resultsin activation of PI3 kinase, PI3K inhibitors are an attractiveoption to consider for potentiation of immunotherapy inPTEN-mutant cancers. Because PI3 kinase is also import-ant for T cell activation, and in fact this represents a majorsignaling pathway regulated by CTLA-4 and PD-1, carefuldrug selection and intermittent scheduling are importantconsiderations [34]. A pan-PI3K inhibitor was shown toblock T cell activation in vivo, whereas a β-isoform-specificinhibitor was shown to improve cancer immunotherapyefficacy in a mouse model [18].The present study has notable limitations. It describes

results with only two patients, and thus additional studiesinvolving a greater sample size will be required to deter-mine the frequency of active β-catenin signaling or PTEN-deletion in tumor cells among cases of secondary immuneresistance. Additionally, due to limited availability ofbiopsy tissue at each time point, not all assays (geneexpression profiling, multiplex immunofluorescence, ge-nomic sequencing) could be performed on all samples foreach patient. Nevertheless, this study provides provocativeexamples of secondary resistance linked to loss of a T cell-inflamed tumor microenvironment.

ConclusionWe report two cases of secondary immune resistance inmetastatic melanoma patients, associated with tumorcell acquisition of either active β-catenin signaling orPTEN gene deletion, two oncogenic aberrations linkedto ineffective T cell infiltration into tumor sites. Our re-sults suggest that acquired alterations in oncogenic sig-naling can be added to the list of mechanisms leadingto tumor outgrowth in the face of immune selectivepressure catalyzed by immunotherapeutic interventions.As the number of patients treated with checkpoint in-hibitors and other immunotherapies continues to grow,and as follow-up time continues to increase, it is likelythat numerous additional secondary resistance caseswill be identified. Such patients should be interrogatedfrom multiple perspectives for novel mechanisms ofimmune escape. As these mechanisms continue to becatalogued, it is hoped that patterns will emerge andnew therapies can be developed to overcome resistanceclinically.

Fig. 4 Loss of PTEN protein expression by melanoma cellsassociated with a lack of CD8+ T cell infiltration. aImmunofluorescence demonstrates that the on-treatment specimenshows PTEN protein expression by SOX10-positive melanoma cellsand CD8+ T cell infiltration (left panels); (b) the therapeutic-resistantpost-treatment specimen (right panels) from the same patientshows minimal PTEN protein expression by SOX10-positivemelanoma cells and no CD8+ T cell infiltration. Multipleximmunofluorescence staining was performed for DAPI, Sox10, CD8,and PTEN; each stain shown separately and merged

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Supplementary informationSupplementary information accompanies this paper at https://doi.org/10.1186/s40425-019-0780-0.

Additional file 1: Table S1. Genes analyzed by the OncoScreen ST2.0next-generation genomic sequencing assay. Table S2. Genes analyzed bythe OncoPlus next-generation genomic sequencing assay.

AbbreviationsAb: Antibody; B2M: Beta-2-microglobulin; BATF3: Basic leucine zipper ATF-liketranscription factor 3; CRISPR: Clustered regularly interspaced shortpalindromic repeats; CTLA-4: Cytotoxic T-lymphocyte-associated protein-4;FFPE: formalin-fixed, paraffin-embedded; H&E: Hematoxylin and eosin;HIPAA: Health Insurance Portability and Accountability Act; HLA-A2: Humanleukocyte Antigen-A2; HTRC: Human Tissue Resource Center;IF: Immunofluorescence; IFNGR1: Interferon-gamma receptor 1; IFN-γ: Interferon-gamma; IgG: Immunoglobulin G; IHC: Immunohistochemistry;MHC: Major histocompatibility complex; NGS: Next-generation genomicsequencing; PBMC: Peripheral blood mononuclear cells; PD-1: Programmedcell death protein-1; PI3K: Phosphatidylinositol 3-kinase; PTEN: Phosphataseand tensin homolog; RECIST 1.0: Response Evaluation Criteria in Solid Tumorsguideline version 1.0; rhIL-2: Recombinant human interleukin-2; TBST: Tris-buffered saline with Tween 20; TCGA: The Cancer Genome Atlas

AcknowledgmentsThe authors would like to thank the patients presented in the study, and thetechnical staff from the Human Immunologic Monitoring facility of theUniversity of Chicago Comprehensive Cancer Center.

Authors’ contributionsTFG and JAT contributed to clinical care. TFG and KM contributed to sampleprocurement. TFG supervised the project. TFG, JL, JAT, YZ, JPS, LR, SSperformed data analysis and interpretation. JPS and LR performed next-generation genomic sequencing and analysis. YZ performed gene expressionprofiling and immunoassays. JAT, JL, and TFG wrote the manuscript with in-put from all other authors. Final approval of submitted manuscript: all au-thors. Accountable for all aspects of work: all authors.

FundingJAT is supported by Elliot Sigal Immuno-oncology Fellowship Research Fund,and NIH 2T32CA009566–31. SS was supported by R00CA204595 transition toindependence award by the NCI. This work was supported by R35CA210098and the American Cancer Society Jules L. Plangere Jr. Family Foundation Pro-fessorship in Cancer Immunotherapy.

Availability of data and materialsThe data sets generated and analyzed during the current study availablefrom the corresponding author on reasonable request.

Ethics approval and consent to participateBiospecimens were obtained from patients who consented to participate ina biobank protocol to collect patient samples for future research. TheInstitutional Review Board of the University of Chicago approved thebiobank protocol.

Consent for publicationParticipants of the biobank protocol provide consent for data generated tobe used in medical and scientific publications Patient names and personalidentifying information are not disclosed and are removed before the useand publication of data.

Competing interestsTFG: has received consultancy fees from Merck, Roche-Genentech, Abbvie,Bayer, Jounce, Aduro, Fog Pharma, Adaptimmune, FivePrime, and Sanofi.T.F.G. has received research support from Roche-Genentech, BMS, Merck,Incyte, Seattle Genetics, Celldex, Ono, Evelo, Bayer, Aduro. T.F.G. has intellec-tual property/licensing agreements with Aduro, Evelo, and BMS. T.F.G is a co-founder/shareholder with Jounce and Pyxis Oncology.JL: DSMB: TTC Oncology; SAB: 7 Hills, Actym, Alphamab Oncology, Array,BeneVir, Mavu, Tempest; Consultancy: Aduro, Astellas, AstraZeneca, Bayer,

Bristol-Myers Squibb, Castle, CheckMate, Compugen, EMD Serono, IDEAYA,Immunocore, Janssen, Jounce, Merck, NewLink, Novartis, RefleXion, SpringBank, Syndax, Tempest, Vividion, WntRx; Research Support: (clinical trials un-less noted) AbbVie, Array (Scientific Research Agreement; SRA), Boston Bio-medical, Bristol-Myers Squibb, Celldex, CheckMate (SRA), Compugen, Corvus,EMD Serono, Evelo (SRA), Delcath, Five Prime, FLX Bio, Genentech, Immuno-core, Incyte, Leap, MedImmune, Macrogenics, Novartis, Pharmacyclics, Palleon(SRA), Merck, Tesaro, Xencor. Travel: Array, AstraZeneca, Bayer, BeneVir,Bristol-Myers Squibb, Castle, CheckMate, EMD Serono, IDEAYA, Immunocore,Janssen, Jounce, Merck, NewLink, Novartis, RefleXion Patents: (bothprovisional) Serial #15/612,657 (Cancer Immunotherapy), PCT/US18/36052(Microbiome Biomarkers for Anti-PD-1/PD-L1 Responsiveness: Diagnostic,Prognostic and Therapeutic Uses Thereof).SS: holds a patent on Wnt/β-catenin targeting to enhance anti-tumor im-mune responses (PCT15/155,099), serves on the SAB on Venn Therapeutics,Tango Therapeutics and consults for TAKEDA, Replimune, Ribon, Torque andArcus.JPS: Honoraria: Bristol-Myers Squibb, AbbVie; Research Funding: AbbVie.LR: Honoraria: Bristol-Myers Squibb, AbbVie; Research Funding: AbbVie; Travel,Accommodations, Expenses: Bristol-Myers Squibb.JAT: declares no competing interests.YZ: declares no competing interests.KM: declares no competing interest.

Author details1Department of Medicine, University of Chicago, 5841 S. Maryland Ave,MC2115, Chicago, IL 60637, USA. 2Department of Pathology, University ofChicago, 5841 S. Maryland Ave, MC2115, Chicago, IL 60637, USA. 3Presentaddress: Koch Institute for Integrative Cancer Research at MassachusettsInstitute of Technology, Department of Biology at MIT, Cambridge, USA.

Received: 17 June 2019 Accepted: 22 October 2019

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